CN113552345B - Exosome quantitative detection method based on immunofluorescence enhancement - Google Patents
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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Abstract
The invention discloses an exosome quantitative detection method based on immunofluorescence enhancement. The method comprises the following steps: extracting exosomes by centrifugation or diluting body fluid to be tested such as blood; and combining the metal nanoparticle fluorescent probe with the exosome or diluted body fluid to be detected, and then placing the metal nanoparticle fluorescent probe on a metal nano film for fluorescent imaging to realize quantitative detection of the exosome or the body fluid to be detected. The invention establishes a fluorescence detection method based on immune enhancement, obtains an enhanced fluorescence signal of exosomes or exosomes with specific sources, establishes a standard curve, obtains the concentration of exosomes to be detected, and can meet the requirements of laboratory and clinical quantitative detection of exosomes and different exosome subgroups.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to an exosome quantitative detection method based on immunofluorescence enhancement.
Background
Exosomes (exosomes) are membrane vesicles with the diameter of 30-100nm which are released into an extracellular matrix after the fusion of intracellular multivesicles and cell membranes, are rich in content and carry proteins, lipids, miRNAs and the like. Exosomes can be released by various types of cells of the body and widely distributed in body fluids such as blood, urine, saliva, milk, interstitial fluid and the like. As a molecular medium, exosomes can promote information communication among cells or organs, participate in various physiological and pathological processes, and are closely related to occurrence, development and progress of diseases. The exosomes reflect the function of the cells of origin, and their content components differ from the healthy state under pathological conditions. Exosomes and exosomes of different origins have different specific surface marker proteins, such as ALIX, annexin, ANXA5, CD171, CD31, CD44, CD63, CD81, CD9, claudins, epCAM, flow 1, GM130, HLA-G, HSP5, HSP70, HSP90, ICAM-1, intersins, rab5, snap, tim, tsg101, etc. Depending on the surface marker proteins, the exosomes may be divided into different sub-populations, or referred to as exosomes of different origin (e.g. tumor origin, nerve origin, placenta origin, etc.). The identification and quantification of exosomes and different exosome subpopulations can provide information for disease prediction, diagnosis, efficacy assessment and prognosis. In the evaluation of the separation and purification methods and separation effects of exosomes (total exosomes, exosomes of different subgroups), it is also necessary to quantify the isolated exosomes.
The method for quantitative detection of exosomes mainly comprises enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western blot), microfluidics, mass spectrometry and the like. Exosomes, particularly exosomes of different subgroups, are low in content in body fluid, and a rapid, simple, convenient, high-sensitivity, economical and applicable detection method is not available at present. Immunofluorescence is an important method in biomedical detection, but the existing method has the defects of low fluorescence brightness, easy quenching and the like.
Disclosure of Invention
In view of the shortcomings of the prior art, the invention aims to provide an exosome quantitative detection method based on immunofluorescence enhancement, which aims to solve the problem of low fluorescence brightness in the existing immunofluorescence method.
The technical scheme of the invention is as follows:
an exosome quantitative detection method based on immunofluorescence enhancement, which comprises the following steps:
extracting exosomes by centrifugation or diluting body fluid to be tested such as blood;
and combining the metal nanoparticle fluorescent probe with the exosome or diluted body fluid to be detected, and then placing the metal nanoparticle fluorescent probe on a metal nano film for fluorescent imaging to realize quantitative detection of the exosome or the body fluid to be detected.
Optionally, the metal nanoparticle fluorescent probe is formed by combining a metal nanorods (GNRs), an exosome surface marker protein antibody and a fluorescent agent.
Optionally, the metal nanorods are gold nanorods.
Alternatively, the exocrine marker antibody is selected from one of an anti-ALIX antibody, an anti-Annexin antibody, an anti-ANXA 5 antibody, an anti-CD 171 antibody, an anti-CD 31 antibody, an anti-CD 44 antibody, an anti-CD 63 antibody, an anti-CD 81 antibody, an anti-CD 9 antibody, an anti-Claudins antibody, an anti-EpCAM antibody, an anti-FLOT 1 antibody, an anti-GM 130 antibody, an anti-HLA-G antibody, an anti-HSP 5 antibody, an anti-HSP 70 antibody, an anti-HSP 90 antibody, an anti-ICAM-1 antibody, an anti-intelins antibody, an anti-RAB 5 antibody, an anti-SNAP antibody, an anti-TIM antibody, an anti-TSG 101 antibody, and the like, including monoclonal or polyclonal antibodies.
Optionally, the fluorescent agent is an AF647 dye.
Optionally, the metal nano film is a nano silver film;
or the metal nano film is a composite film formed by a nano silver film and a PC668 photon crystal film formed on the nano silver film;
or, the metal nano film is a nano gold film.
The beneficial effects are that: the invention establishes a fluorescence detection method based on immune enhancement, obtains an enhanced fluorescence signal of exosomes or exosomes with specific sources, establishes a standard curve, obtains the concentration of exosomes to be detected, and can meet the requirements of laboratory and clinical quantitative detection of exosomes and different exosome subgroups. By the method, the sensitivity of quantitative detection of exosomes is remarkably improved.
Drawings
FIG. 1 is a schematic diagram of gold nanorod coated antibodies and a "sandwich" immunoassay exosome. Take the example of CD63 surface markings.
FIG. 2 is a surface morphology feature of GNRs/Ag (silver) substrates for fluorescence enhancement; wherein a) is a transmission electron microscope image of GNRs. b) Images of solution of GNRs (λ=645 nm) and antibody-coated GNRs (λ=662 nm) under uv-ir spectra. c) Fluorescent images of AF647 molecules on GNRs/Ag substrates, GNRs, silver island films and glass substrates, respectively. d) A histogram of 4 basal fluorescence enhancement factors in c).
FIG. 3 is a graph showing the results of microarray detection of human CD63 surface-labeled exosomes on a GNRs/Ag substrate. a) CD63 surface-labeled exosomes were diluted to different concentrations for detection, and each sample was repeated 4 times. The upper left of the graph shows a control, and the remaining concentrations are, in turn, 500-0.4ng/mL. b) CD63 surface-labeled exosome detection standard curve.
FIG. 4 is a schematic diagram showing the results of detecting exosomes (CD 63 surface markers) in plasma of patients with liver fibrosis and liver cancer. a) For scanning fluorescent images generated by microarrays with AF647 fluorescent-labeled antibodies on GNRs/Ag substrates, the upper row of detection results for 2 controls; patients 1-3, patients with liver fibrosis; 4-6, liver cancer patients. b) For detecting liver fibrosis based on GNRs/Ag substrate, the fluorescence intensity histogram of plasma exosomes of liver cancer patients is <0.05.
FIG. 5 is a graph showing fluorescence intensity of AF647 on different substrates; wherein a) is a schematic diagram of fluorescence intensity of AF647 on glass, ag film, PC668, ag-PC668 substrate, respectively, and b) is a histogram of fluorescence intensity of AF647 on glass, ag film, PC668, ag-PC668 substrate, respectively.
Detailed Description
The invention provides an exosome quantitative detection method based on immunofluorescence enhancement, which is used for making the purposes, technical schemes and effects of the invention clearer and more definite, and is further described in detail below. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
The invention provides an exosome quantitative detection method based on immunofluorescence enhancement, which comprises the following steps:
extracting exosomes by centrifugation or diluting body fluid to be tested such as blood;
and combining the metal nanoparticle fluorescent probe with the exosome or diluted body fluid to be detected, and then placing the metal nanoparticle fluorescent probe on a metal nano film for fluorescent imaging to realize quantitative detection of the exosome or the body fluid to be detected.
In one embodiment, the metal nanoparticle fluorescent probe is formed by combining a metal nanorod, an exosome surface marker protein antibody and a fluorescent agent. Further, the metal nanorods are gold nanorods. Further, the exosome marker protein antibody is selected from one of an anti-ALIX antibody, an anti-Annexin antibody, an anti-ANXA 5 antibody, an anti-CD 171 antibody, an anti-CD 31 antibody, an anti-CD 44 antibody, an anti-CD 63 antibody, an anti-CD 81 antibody, an anti-CD 9 antibody, an anti-Claudins antibody, an anti-EpCAM antibody, an anti-FLOT 1 antibody, an anti-GM 130 antibody, an anti-HLA-G antibody, an anti-HSP 5 antibody, an anti-HSP 70 antibody, an anti-HSP 90 antibody, an anti-ICAM-1 antibody, an anti-intelins antibody, an anti-RAB 5 antibody, an anti-SNAP antibody, an anti-TIM antibody, an anti-TSG 101 antibody, and the like, including monoclonal or polyclonal antibodies. Further, the fluorescent agent is AF647 dye.
In one embodiment, the metal nanomembrane is a nanosilver film or an Ag-PC668 or nanogold film.
Referring to fig. 1, the present embodiment provides a quantitative detection method for exosomes based on immunofluorescence enhancement, specifically designs a fluorescence enhancement structure based on a surface plasmon of silver nanoparticles, or designs a fluorescence enhancement structure based on a principle of coupling a surface plasmon of silver nanoparticles with a colloidal photonic crystal, namely a nano silver film or a photonic crystal-nano silver composite film (Ag-PC 668) deposited on a substrate; a fluorescence enhancement structure, i.e. a nano-gold film deposited on a substrate, can also be designed based on gold nanoparticle surface plasmons. Meanwhile, a sandwich immunoassay method based on gold nanorods is established to quantitatively detect exosomes. Gold nanorods are coated with exosome surface marker protein antibodies, such as monoclonal or polyclonal antibodies against ALIX, annexin, ANXA5, CD171, CD31, CD44, CD63, CD81, CD9, claudins, epCAM, FLOT1, GM130, HLA-G, HSP5, HSP70, HSP90, ICAM-1, integrins, RAB5, SNAP, TIM, TSG101, and the like. Extracting total exosomes from body fluid (plasma, serum, urine, tissue fluid), cells and tissue culture fluid, and specifically capturing exosomes or exosome subpopulations with corresponding surface marker proteins by using gold nanorods coated with antibodies. Once the antigen is captured by the antibody-coated gold nanorods, another type of specific primary antibody will be used to bind the antigen. The secondary antibody with fluorescent label (AF 647) was used to identify the primary antibody for binding to antigen. The gold nanorod suspension with the fluorescent mark attached is dripped on a nano silver film or Ag-PC668 or nano gold film substrate, and is excited by laser (such as 635nm laser) to form images in a microarray scanner. And (3) constructing a standard curve by taking exosome specific surface marker proteins or exosomes with different concentrations as standard substances to detect, so as to obtain the corresponding concentration of the exosomes to be detected. By the method, the sensitivity of quantitative detection of exosomes is remarkably improved, compared with a glass substrate, 360 times of enhancement is obtained when a nano silver film is used as a substrate, and higher enhancement times are obtained when Ag-PC668 or a nano gold film is used as a substrate. And the method can detect more than 4 patients at a time, and each patient is repeated 3 times.
The invention is further illustrated by the following specific examples.
Example 1
1. Preparation of nano silver film (silver island film):
a. the cleaned glass slide is put into a prepared piranha solution (the hydrogen peroxide and the concentrated sulfuric acid are mixed according to the volume ratio of 1:3) and soaked for 8 hours.
b. The soaked slide was placed in a solution containing 45mL of 0.03M AgNO 3 To the petri dish of the solution, 3mL of ammonia water was added and the mixture was shaken in a shaker for 30min.
c. Placing the glass slide treated in the step b into 60mL of 0.04M AgNO 3 The solution was mixed with 30ml of 1.25M NaOH solution. The precipitate formed was redissolved by adding 12mL of aqueous ammonia. Subsequently, a glucose solution having a volume of 30mL and a concentration of 0.25M was added to reduce silver ions to silver metal, and a silver film was formed on the glass slide. After 20min, the slide glass was taken out of the petri dish, rinsed with ultrapure water, dried with nitrogen, and stored in a sealed light-proof manner.
2. Preparation and functionalization of gold nanorods
a. Preparation of gold nanorods
Gold nanorods are prepared by a two-step seed-mediated growth method at room temperature, and the specific steps are as follows:
first, 5mL of a 0.2M CTAB (cetyltrimethylammonium bromide) solution was mixed with 5mL of 5X 10 -4 HAuCl of M 4 After mixing the solutions and gentle stirring, freshly prepared chilled 0.6ml0.01m NaBH was added 4 The solution (water as solvent) was stirred for 2min to turn the color of the solution brown yellow to obtain a seed solution.
Then, 0.2mL of 0.04M AgNO was added to 5mL of 0.2M CTAB solution 3 The solution was then added with 5mL of 0.001M HAuCl 4 The solution was gently mixed so that the solution became golden yellow. 0.07ml of a 0.0788M ascorbic acid solution was added, and the mixture was shaken well to turn the solution colorless, thereby obtaining a growth solution.
Finally, 20. Mu.L of seed solution was added to the growth solution, and the mixture was left overnight at room temperature. And when the growth solution is deep magenta, the gold nanorods are successfully formed. 1mL of the above overnight solution was centrifuged at 9600rpm three times for 20min each. After the third round, the gold nanorod pellet was re-dispersed in water to remove excess CTAB and spherical nanoparticles. The gold nanorod precipitate was dispersed in 50. Mu.L of water for storage.
b. Functionalization of gold nanorods
The gold nanorod solution was immersed in ethanol containing 20mM MUA (11-mercaptoundecanoic acid) overnight at room temperature. The MUA-modified gold nanorods were collected by centrifugation at 14,000rpm for 12min, washed with ethanol and dried, and then immersed in a methanol solution containing 20mM 1-ethyl-3- (-3-dimethylaminopropyl carbodiimide hydrochloride (EDC) and 20mM n-hydroxysuccinimide (NHS) for 30min to activate carboxyl groups for antibody coupling.
3. Detection of CD63 surface marker exosome of liver fibrosis and liver cancer patient plasma
a. Drawing of a Standard Curve
Carboxyl group functionalized gold nanorods were used to coat primary antibodies (i.e., rabbit anti-exosome specific marker protein monoclonal anti-CD 63 antibodies) and placed in PBS (phosphate buffer) with a mass concentration of 15% fbs (fetal bovine serum), diluted 1/100, and shaken in a shaker for 2h. Washed 3 times with PBS. The control replaced primary antibody with pure FBS. Standard proteins were diluted with PBS containing 15% FBS at final concentrations of 0.4-500ng/mL. 50 μl was added to the gold nanorods, incubated for 2h in a shaker, and the gold nanorods were washed 3 times with PBS. The mouse anti-exosome specific marker protein monoclonal antibody is added, and incubated for 1h in a shaker at room temperature. Washing 3 times with PBS, adding AF 647-labeled goat anti-mouse antibody, incubating for 30min at room temperature under shaking in the absence of light, and washing 3 times with PBS. The gold nanorod suspension with the fluorescent label attached is dripped on a nano silver film or an Ag-PC668 substrate or a nano gold film, and imaged in a microarray scanner under a 635nm laser channel. After the fluorescence intensity of the emitted light is obtained, a standard curve is drawn.
FIG. 2 is a surface morphology characterization of GNRs/Ag substrates for fluorescence enhancement. In fig. 2 a) shows that GNRs are uniform in size. B) in fig. 2 is an image of GNRs (λ=645 nm) and antibody-coated GNRs (λ=662 nm) solutions in the uv-ir spectrum. FIG. 2 c) is a fluorescent image of AF647 molecules on GNRs/Ag substrate, GNRs, silver island film and glass, respectively. D) in FIG. 2 is the direct square of the 4 basal fluorescence enhancement factors in c).
b. Quantitative detection of exosomes in plasma of liver fibrosis and liver cancer patient
Extracting exosomes from the plasma of patients with liver fibrosis and liver cancer by using a centrifugal method, or diluting a human plasma sample, coating a gold nanorod with a rabbit anti-human anti-CD 63 antibody, capturing exosomes to be quantified from the exosomes or diluted plasma, and detecting surface enhanced immunofluorescence to obtain the fluorescence intensity of the exosomes in the plasma of patients with tumor, which is enhanced compared with that of a contrast. The concentration of patient plasma exosomes was obtained by a standard curve (fig. 3). Specifically, FIG. 4 a) is a fluorescent image generated by scanning a microarray of GNRs/Ag substrates with AF647 fluorescent-labeled antibodies, where the upper row is the detection result of 2 healthy controls; the following three rows are patient test results: patients 1-3, patients with liver fibrosis; 4-6, liver cancer patients. In fig. 4 b) is a fluorescence intensity histogram of plasma exosomes of controls and cases (liver fibrosis, liver cancer patients) based on GNRs/Ag basal detection. CD63 is used as a standard substance to make a standard curve (figure 3), and the concentrations of exosomes in the plasma of 3 liver fibrosis patients are respectively 16,24 and 28ng/mL, and the concentrations of liver cancer patients are respectively 34,44 and 54ng/mL. Depending on the throughput of the microarray, the method can be used to simultaneously test at least 4 or more patients at a time, and each patient is repeated 3 times.
4. Preparation of photon crystal and silver nano film complex and comparison of fluorescence enhancement effects of different substrates
a. Synthesis of p (St-co-NIPAM) (Poly (styrene-isopropyl acrylamide)) microspheres
1.54g of NIPAM and 0.3g of BIS (N, N-methylenebisacrylamide) monomer were dissolved in 95mL of ultrapure water and then charged into a three-necked round bottom flask equipped with a condenser, thermometer and gas purge inlet. The solution in the flask was heated to 70 ℃ while stirring, while being purged with nitrogen for at least 30min. About 5 minutes before initiation, 6.6g of styrene monomer was added, and ammonium persulfate was added to initiate the reaction. Stirring is carried out for 24h under nitrogen. Filtration using Whatman filter paper (No. 2) to remove coagulum, centrifugation removed all unreacted monomer, yielded photonic crystal p (St-NIPAM) microspheres with a particle size of 668nm.
b. Preparation of photonic crystal thin films
The glass substrate was first treated with a chromium sulfate solution to ensure surface cleaning, and then vertically placed in a vial containing approximately 0.2wt% of a monodisperse p (St-NIPAM) aqueous suspension at a constant temperature (50 ℃) and humidity (60%) for 48 hours, and a photonic crystal film (PC) was deposited on the glass substrate. The photonic crystal film (photonic crystal particle diameter: 668 nm) was a violet photonic crystal film, designated as PC668.
c. Preparation of complex composed of photonic crystal and nano silver
And covering the PC668 on the silver island film to obtain a complex formed by the photonic crystal film and the nano silver film, which is named as Ag-PC668.
d. Comparison of fluorescence enhancement effects of different substrates
The fluorescence intensity characteristics of the fluorescent dye AF647 molecules were measured using Ag-PC668 and nano-silver film (noted Ag), glass slide (glass), and PC668 alone as substrates, respectively. From the results in fig. 5 a), it is shown that the fluorescence intensity of AF647 on the nano-silver film, PC668 and Ag-PC668 is significantly increased as compared to the glass slide. And the fluorescence intensity of AF647 is strongest when Ag-PC668 is used as a substrate, as shown in FIG. 5 a). After subtraction of the blank background, the AF647 molecular fluorescence intensity histogram is shown in fig. 5 b), which shows the fold increase of fluorescence for the different substrates. The results show that the fold increase in fluorescence is highest with Ag-PC668 as the substrate. Therefore, when the concentration of exosomes is quantitatively detected, ag-PC668 can be used as a substrate, and the Ag-PC668 and the gold nanorods form sandwich immunofluorescence detection, so that the effect is better.
5. Preparation of plasma gold film on glass slide
The slide was immersed in HAuCl at a concentration of 3mM 4 In solution. mu.L ammonium hydroxide (NH) was added per ml total volume 4 OH), oscillation 1And (5) min. After rinsing with deionized water, the slide was immersed in sodium borohydride (NaBH) at a concentration of 1mM 4 ) The solution was kept for 1min. The slide was rinsed again with deionized water and the substrate was immersed in 1:1 HAuCl 4 And NH 2 In OH aqueous solution (750. Mu.M), at room temperature, shake for 15-20min. The slide glass is then washed with deionized water, dried after washing, and stored in a sealed container for use.
It is to be understood that the invention is not limited in its application to the examples described above, but is capable of modification and variation in light of the above teachings by those skilled in the art, and that all such modifications and variations are intended to be included within the scope of the appended claims.
Claims (1)
1. An exosome quantitative detection method based on immunofluorescence enhancement is characterized by comprising the following steps:
extracting exosomes by adopting a centrifugal method or diluting body fluid to be tested;
combining a metal nanoparticle fluorescent probe with an exosome or diluted body fluid to be detected, then placing the metal nanoparticle fluorescent probe on a metal nano film for fluorescent imaging, and constructing a standard curve by taking exosome specific surface marker proteins or exosomes with different concentrations as standard substances for detection to obtain the corresponding concentration of the exosomes to be detected, so as to realize quantitative detection of the exosomes in the exosomes or the body fluid to be detected;
the metal nanoparticle fluorescent probe is formed by combining a metal nanorod, an exosome surface marker protein antibody and a fluorescent agent, wherein the metal nanorod is a gold nanorod, the exosome surface marker protein antibody is selected from one of an anti-ALIX antibody, an anti-Annexin antibody, an anti-ANXA 5 antibody, an anti-CD 171 antibody, an anti-CD 31 antibody, an anti-CD 44 antibody, an anti-CD 63 antibody, an anti-CD 81 antibody, an anti-CD 9 antibody, an anti-Claudins antibody, an anti-EpCAM antibody, an anti-FLOT 1 antibody, an anti-GM 130 antibody, an anti-HLA-G antibody, an anti-HSP 5 antibody, an anti-HSP 70 antibody, an anti-HSP 90 antibody, an anti-ICAM-1 antibody, an anti-Integrins antibody, an anti-RAB 5 antibody, an anti-SNAP antibody, an anti-TIM antibody and an anti-TSG 101 antibody, and the fluorescent agent is an AF647 dye;
the metal nano film is a composite film formed by a nano silver film and a PC668 photon crystal film formed on the nano silver film;
the PC668 photonic crystal film is prepared from poly (styrene-isopropyl acrylamide) microspheres;
the photonic crystal particle size in the PC668 photonic crystal film is 668nm.
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